Plants provide an almost endless variety of chemical compounds derived from primary or secondary metabolism. Many plant secondary metabolites are desirable. For example, some plant secondary metabolites provide protection against pathogens or adverse environmental conditions, and thus have substantial agronomic importance. In addition, a number of plant secondary metabolites serve as nutraceutical components of our diet. Furthermore, certain plant secondary metabolites have diverse medical applications, particularly in the pharmaceutical industry (See, Heilmann J. and R. Bauer (1999) Functions of Plant Secondary Metabolites and their Exploitation in Biotechnology. M. Wink. Boca Raton, CRC Press LLC. 3:274–310).
The accumulation of certain secondary metabolites in plants, however, can also be problematic. For example, the presence in trees of large amounts of lignin, a product of the plant phenylpropanoid pathway, can increase the costs and time required to make high quality paper. Large amounts of lignin in grasses can decrease their digestibility. In flour products, high levels of colored pigments, which are also products of the phenylpropanoid pathway, can make the flour products less desirable to the consumer.
Plant secondary metabolites can be grouped into several major classes including the phenolics, alkaloids, and isoprenoids. The amino acids phenylalanine and tyrosine serve as precursors for phenolic compounds that are intermediates or final products of a branch of the phenylpropanoid pathway. A schematic representation of the plenylpropanoid pathway which leads from phenylalanine through several branches to the hydroxy cinnamates, lignins, and the flavonoids is shown in FIG. 1. The phenylpropanoids, and their derivatives, and the flavonoids, and their derivatives, are examples of intermediates and final products of the phenylpropanoid pathway respectively. Flavonoids are phenolic natural products that have multiple functions in plants, including roles as floral pigments for the attraction of pollinators, signaling molecules for beneficial microorganisms in the rhizosphere, and antimicrobial defense compounds. In addition, flavonoids are emerging as important nutraceuticals because of their strong antioxidant properties, and several flavonoids show anti-tumor activities. Chlorogenic acid, another phenolic compound that is believed to be the final product of one branch of the phenylpropanoid pathway has anti-pathogenic activity and bactericidal activity in plant and anti-tumor activity in animals.
The first committed step in the phenylpropanoid pathway is catalyzed by phenylalanine ammonia lyase (PAL), which converts phenylalanine to cinnamic acid (or tyrosine to ρ-coumaric acid in some monocots). Transcriptional activation of genes encoding enzymes involved in phenylpropanoid metabolism, such as PAL, 4-coumarate CoA ligase (4CL), and cinnamyl alcohol dehydrogenase (CAD), represents a key step in the regulation of the phenylpropanoid pathway. The coordinate regulation of the PAL, 4CL and CAD genes in many plant species suggests the existence of specific transcription factors or transactivators that coordinately activate the expression of these genes.
The regulation of flavonoid biosynthesis provides the best described example of how certain transcription factors control the expression of biosynthetic genes (reviewed in Mol et al. 41). In maize, two classes of regulatory proteins control accumulation of the anthocyanins which are flavonoid derivatives. These two classes are a Myb-domain containing class (encoded by the CI and P1 genes) and a basic helix-loop-helix (bHLH)-domain containing class (members of the RIB gene families). Anthocyanin production requires the interaction between a member of the Myb-domain C1/P1 family and a member of the bHLH-domain R/B family 41, and the pattern of anthocyanin pigmentation in any particular plant part is controlled by the combinatorial, tissue-specific expression of these regulatory genes. Orthologs, as defined by Fitch, of the maize C1 and R regulators have been identified in other plants, such as petunia and snapdragon, and these regulatory proteins have been shown to be exchangeable between monocots and dicots.
In addition to 3-hydroxy flavonoids and anthocyanins, maize and its close relatives like sorghum accumulate 3-deoxy flavonoids and derived pigments, which include the phlobaphenes. A single known transcription factor (P) controls 3-deoxy flavonoid and phlobaphene biosynthesis in maize. P regulates the accumulation of a subset of flavonoid biosynthetic gene products, namely C2 (a chalcone synthase) and A1 (dihydroflavonol 4-reductase). On the basis of these and other studies, it is quite clear that transcription factors are important tools for controlling the levels of flavonoids in plants.
In view of the important role of phenolic compounds that are intermediates and final products of the plant phenylpropanoid pathway, it is desirable to have additional transcription factors which are capable of regulating the levels of these secondary metabolites in plants. Such transactivators would serve as important tools for increasing pathogen resistance, altering digestibility, and manipulating levels of nutraceutical compounds, such as flavonoids and other phenolic compounds, in plants.